Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives.
A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).
Miller
E J
EJ
Department of Biochemistry, University of Alabama, Birmingham 35294.
Narkates
A J
AJ
Niemann
M A
MA
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002138:Calibration; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003094:Collagen / Q000032:analysis* / Q000378:metabolism; D003553:Cystine / Q000737:chemistry / Q000378:metabolism; D005449:Fluorenes / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D006901:Hydroxylysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D005449:Fluorenes; C022819:hydroxylysine glycoside; D006901:Hydroxylysine; D014443:Tyrosine; D003553:Cystine; D006639:Histidine; D003094:Collagen; C054007:1-(9-fluorenyl)methyl chloroformate